The overall objective of the proposed research is an elucidation of the mechanisms by which low molecular weight ligands alter and thus potentially regulate the enzymatic activities of the aspartokinase I- homoserine dehydrogenase I complex of E. coli K12. The enzyme complex consists of four apparently identical subunits at high enzyme concentration; however, the complex does dissociate to form smaller species under some conditions and one of our first objectives will be to investigate the role that this reversible subunit association- dissociation equilibrium plays in determining the kinetic and regulatory properties of the enzyme. Direct optical scanning of stacked gel chromatographic columns using the equilibrium saturation technique will be employed to evaluate the stoichiometries and equilibrium constants of the equilibria involved as well as to determine the effects of pH, substrates, and modifying ligands on these equilibria. In addition, the effects of these substrates and ligands on the conformation of the protein will be investigated by fluorescence and other optical techniques. A correlation of these results with those obtained by kinetic and direct binding studies should contribute significantly to our understanding of the regulatory mechanisms of this enzyme complex.